Products and methods for single parameter and multiparameter phenotyping of cells

ABSTRACT

A method of single parameter and multiparameter characterizing of cells, particularly immunophenotyping of cells, is provided. The method preferably uses antibody-coated microspheres which are adapted to bind to specific types of cells. One or more sets of coated microspheres are contacted simultaneously or sequentially with a suspension of cells and bind the cells they are adapted to bind to form bead-cell complexes. Cells may bind to one or more microspheres. The bead-cell complexes are then separated from the suspension The complexes are preferably stained and then examined to characterize the cells, preferably the cells bound to the microspheres. A method of quantitating a specific cell type is provided. A kit and apparatus for performing the method are also provided.

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/563,564 filed May 3, 2000. This application claims thebenefit of U.S. Provisional Application Serial No. 60/132,395 filed May4, 1999. The contents of application Ser. Nos. 09/563,564 and 60/132,395are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to phenotyping andimmunophenotyping of cells and more particularly to single parameter andmultiparameter phenotyping and immunophenotyping of cells.

BACKGROUND OF THE INVENTION

Immunophenotyping of cells and tumors, particularly hematopoietictumors, is often of critical importance for clinical evaluation ofcancer patients. However, currently available methodologies,particularly flow cytometry, are expensive and require a high degree ofsuspicion at the time of biopsy. All too often, even before thediagnosis of cancer is made, precious tissue must be set aside forpossible immunophenotyping. If tissue is not set aside and there iscancer present, the correct subtyping of the tumor (and properassignment to treatment protocols) cannot be done after the fact.Methods that do not require forethought, such as immunostaining ofparaffin blocks, are far less sensitive and do not work well inlaboratories that do not perform these stains frequently. Flow cytometryis the currently accepted “gold standard” for immunophenotyping ofhematopoietic cell types. However, there are several problems with themethod. The expense of establishing and maintaining these laboratoriesis perhaps the most severe problem. Generally large hospitals, academiccenters, or commercial reference laboratories are the only institutionscapable of establishing flow cytometry laboratories. These laboratoriesoften charge a premium for their services, and transportation ofspecimens to laboratories is not a trivial problem. Since flow cytometryrequires live cells, specimens must be handled under sterile conditions.In laboratories where the technology is unavailable, a fresh specimenhas to be prepared and shipped to a flow cytometry laboratory understerile conditions for evaluation. Uncontrollable factors such astemperature variations, rough handling, bacterial contamination, orshipping delays may render samples unsuitable for analysis. In addition,flow cytometry requires technologists who have specialized training andtheir time must often be dedicated solely to the technology itself,further increasing the expense of the procedure. Relatively largevolumes of cells must be analyzed in order to obtain statisticallymeaningful results during analysis. In addition, red cells must beremoved from the sample prior to analysis. This is because the number ofred cells in blood and bone marrow samples is far greater than othercells types, and shear numbers alone would overwhelm the sensitivedetectors of the machines. The sample preparation method thereforerequires Ficoll-Hypaque separation, followed by multiple washes,followed by a lysis step to lyse remaining red cells. This methodvirtually eliminates megakaryocytes from most analysis and frequentlydestroys delicate malignant cells (particularly from the relativelycommon tumors such as large cell lymphoma and Hodgkin's disease). It isin these situations that the great sensitivity and complexity of flowcytometry may work to its disadvantage.

Despite the problems described above, however, flow cytometry can veryaccurately and with great sensitivity identify the presence of malignantcells and characterize the kind of malignant cells. Without theinformation that flow cytometry provides, cases can be frequentlyincorrectly diagnosed with catastrophic consequences for the patient.This is particularly true in the setting of a type of biopsy called fineneedle aspiration where examination of a slide alone by light microscopymay be quite difficult.

What would be very useful to the average hospital pathologist or to anyphysician in an outpatient or remote setting is a device or kit thatwould allow the same kind of single parameter or multiparameter analysisof samples using cheaper, more readily available materials. This wouldeliminate the need for specialized laboratories and technologistsdedicated solely to the flow cytometry technology itself and would allowany well trained clinical laboratorian ready access to the same kind ofanalysis. Furthermore, if the need for live cells could be eliminated,cells could be preserved by appropriate fixatives which would broadenthe availability of immunophenotyping data.

Over the last 20 years there has been a tremendous growth in theidentification and characterization of molecules expressed by bloodcells on their cell membranes (called cell surface antigens). Thisgrowth in understanding has been accompanied by the refinement oftechnologies that allow the rapid and sensitive identification of thesemolecules on the surfaces of live cells. However, the overwhelmingmajority of these cell surface antigens are not unique to one type ofcell. There is only rarely a single diagnostic marker to identify a celltype. Instead, most cell populations must be characterizing by analyzingmultiple parameters at the same time.

Antibodies are proteins produced by the body's immune system that havethe property that they bind to a single site on a specific molecule(referred to as an antigen). Antigen-antibody complexes are formed whenan antibody binds its respective antigen. Normally, these complexes arethen cleared by the immune system to rid the body of an infection.However, the immune system has a virtually limitless capacity to produceunique antibodies, which can be tailored to identify particularsubstances, even when present in very small quantities. Antibodies arenow commercially produced to literally hundreds of different antigens.Furthermore, antibodies can be easily tagged with marker molecules, suchas fluorescent molecules, dyes, or other substances that makeidentifying the presence of an antigen-antibody complex a relativelysimple matter. This well-known biochemical reaction has been used todevelop a methodology called flow cytometry. In flow cytometry, intactcells are treated with antibodies that bind specific markers on the cellsurface. The antibodies are, in turn, labeled with a fluorescentmolecule and the cell suspension then flows past a light beam with alight detector which counts the number of fluorescent cells versus theother cells present. This technology has proved tremendously useful inidentifying malignant cell populations in blood and tissue samples frompatients.

In flow cytometry, a cell suspension is treated with antibodies labeledwith fluorescent molecules (fluorochromes), washed, and placed in themachine. The cell suspension is “focused” using buffer solutions so thatthe cells pass through the flow detector in a single file. When eachcell passes through the flow detector, a beam of laser light is passedthrough the cell. Some of the light passes through the cell (calledforward light scatter) and some is refracted at an angle (called sidescatter). Forward scatter increases with a cell's size and side scatterincreases with a cell's internal complexity (mostly granules within thecytoplasm). Thus using just these two measurements, individual celltypes can be roughly categorized. However, there are also lightdetectors, which, by using appropriate color filters, can specificallydetect the fluorescence given off by the antibodies that are attached tothe cell surface. Since current state of the art machines have up tofour different color detectors (referred to as four-color flowcytometry), up to four different antibodies can be added to the sametube. Samples from individual patients are usually divided into multipletubes, each of which contains multiple antibodies. Data analysis istherefore quite complex, and requires computers that are capable ofsimultaneously displaying multiple plots from each tube. This isreferred to as multiparameter analysis. This simultaneous analysis ofmultiple parameters is necessary to first electronically isolate andthen characterize cell populations. Therefore, even though modem flowcytometers analyze up to 6 simultaneous parameters (forward scatter,side scatter, and four antibodies) 3 of the parameters must be used forelectronic isolation of cell types (forward scatter, side scatter, andCD45 staining intensity). Broad categories of cells present inhematologic samples are known in the art and include myeloid cells,monocytes, lymphocytes, megakaryocytes, and red cells. In other words,these 3 parameters must be used to roughly mimic what the human eye doesso effortlessly: identify or characterize broad categories of cells.Indeed, laboratories commonly hire technicians with 2 years of training(only part of which is in the area of hematology) who can, with a veryreasonable degree of accuracy and precision, identify or characterizedifferent cell types present in blood samples. With some additionaltraining, they can also correctly enumerate cell types within bonemarrow aspirate samples. Thus if the human eye were also equipped withthe means to also identify cell surface antigens, there would be no needfor flow cytometry for this purpose. Furthermore, of the remaining 3parameters available for analysis on the flow cytometry, only 2 can bedisplayed in any one plot although new software exists that can display3 dimensional plots. While 3 dimensional plots add to convenience andare applicable in limited situations, two parameter analysis is quitesufficient in most cases. This last point is critical, since any methodthat seeks to supplant flow cytometry must have the ability tocharacterize at least 2 cell surface markers simultaneously.

Analysis of cell populations by flow cytometry is not a trivial processand requires highly trained personnel as outlined above. Both singleparameter and multiparameter analysis can be performed. If data isanalyzed as histogram plots of fluorescence of a single marker versuscell number, then one parameter analysis is being performed. Analyzingtwo such histograms of a single gated cell population could then bereferred to as simultaneous single parameter analysis. An example ofsimultaneous single parameter analysis would involve the use of suchplots to identify cell surface expression of both the B-cell marker CD20and the light chain kappa. Analysis of the binding of each set ofantibodies is independent of the other. In multiparameter analysis, thebinding of the two antibodies are linked and are not independent.Analytical methods require the binding of both antibodies simultaneouslybrought together in a single histogram such as fluorescence 1 versusfluorescence 2. Characterization of the target cell population is bestperformed by analysis of this fluorescence 1 vs. fluorescence 2 plot andanalyzing the binding characteristics of each of these antibodiestogether. This decreases the possibility of an error that wouldincorrectly analyze two overlapping cell populations as a single cellpopulation.

Finally, with the limited exception of DNA ploidy analysis,characterization of solid tumors and non-hematopoietic tumors is quitelimited by flow cytometry. Often there are not well developed protocolsfor developing cell suspensions. In addition, tumor cells may bedelicate and may not survive processing. In addition, many markers usedfor solid tumors such as vimentin or smooth muscle actin areintracytoplasmic antigens and may be difficult to assay by flowcytometry. In addition, most available markers for these other tumorsare not specific markers for the tumors and many normal cells, includingcells present in the background of the available sample, may be stronglypositive for the same markers. Therefore, interpretation of these kindsof samples without specific morphologic correlation is hazardous atbest.

An object of the invention is to provide a cheaper, more accessiblemethod for single parameter and multiparameter analysis of cellpopulations. This analysis is not limited to just cell surface markersbut also optionally includes identifying active receptor sites on cellsurfaces, loss of cell surface proteins, intracellular proteins, andintracellular nucleic acid sequences. One of the features of thisinvention is that the target cell population is being analyzed bypreserving morphologic characteristics of the cells for analysis. Inaddition, it is also possible to count events to obtain specific cellnumbers in relation to specific sample volume.

SUMMARY OF THE INVENTION

A method of characterizing cells comprising the steps of: a) providing asuspension of cells in a liquid medium, said cells including firstcells, b) contacting a group of first beads with said suspension, eachof said first beads being coated with a binding substance or beingmagnetic such that each first bead is adapted to bind to at least one ofsaid first cells, c) incubating said first beads with said suspensionfor a period of time effective to permit said first cells to bind tosaid first beads to form first bead-first cell complexes, each firstbead-first cell complex comprising a first bead and a first cell, d)separating said first bead-first cell complexes from said suspension,and e) examining said separated first bead-first cell complexes andcharacterizing said first cells. A method of quantitating a specificcell type in a cell suspension is also provided which comprises thesteps of providing a suspension of cells in a liquid medium, countingthe number of certain cells per unit volume of the suspension,incubating the suspension in beads, estimating the relative percentagesof certain cells bound to beads, and calculating the number of certaincells per unit volume of the suspension. A method for preparing anenriched population of a target cell is also provided, which includesthe step of separating first bead-target cell complexes from asuspension and resuspending the target cells in another liquid medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration showing a cell bound to an antibodywhich is bound to or coated on a bead.

FIG. 2 is a schematic illustration showing a number of cells bound to abead.

FIG. 3 is a schematic illustration showing a number of cells bound to abead in the center, and five smaller beads bound to five of the cells.

FIG. 4 is a schematic illustration of an automated device for performingphenotypic or immunophenotypic analysis in accordance with the presentinvention.

FIG. 5. is a schematic illustration of a single slide for use with theautomated device of FIG. 4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

As used herein, when a preferred range such as 5-25 is given, this meanspreferably at least 5 and, separately and independently, preferably notmore than 25. The cells herein are preferably human cells. If a firstgroup of cells does not include members of a second group of cells, andthe second group of cells does not include members of the first group ofcells, the two groups do not overlap. “Visually distinguishable”includes visually distinguishable via light microscopy. Quantitateincludes to estimate or enumerate or count the number of. Phenotypingincludes immunophenotyping and genotyping.

The invention uses beads. As used herein, beads means small particles orsupport surfaces, preferably microspheres, more preferably plasticmicrospheres, more preferably polystyrene microspheres (also referred toas latex beads or spheres or microspheres), which have preferably beencoated with a binding substance or which are magnetic. As used in theclaims, “first beads” includes beads which have been coated with abinding substance or which are magnetic. The bead may be any solidsupport surface or particle that can be suspended in an appropriatesolution. Preferred beads are available as polystyrene microspheres fromBangs Laboratories, Fishers, Ind. The bead sizes are preferably greaterthan 5 microns diameter, preferably 5.5-10.3 microns, less preferably atleast 5.5 or 10 or 12 microns and preferably not more than 12, 15, 20 or30 microns diameter, far less preferably less than 5 microns, such as atleast 1, 2 or 3 microns diameter. 5.5 and 10.3 micron beads arepreferred. The beads can also be colored, such as red or blue, lesspreferably green, purple, orange, brown, yellow, or any other color. Thebeads are preferably coated with a binding substance, such as antibodiesor immunoreactive proteins, or any molecule that can bind to, orinteract with a cell surface in such a way as to bring the cell and thebead into contact or adherence with each other or to bind with eachother; alternatively, the bead can contact or bind with the cell surfacethrough electrostatic charge interactions or magnetic interaction; allof these concepts being covered by the terms “binding to” or “bind to”.When a bead binds to a cell, it forms a bead-cell complex. In theinvention the cell-bead interaction forms a large enough complex toinhibit the passage through a filter containing pores of appropriatedimensions. The filters are preferably sized and selected such thatunbound cells and beads will pass through the pores but bound cell-beadsdo not. These complexes are then transferred to a glass slide andstained with a variety of stains so as to render the complexes visibleby routine microscopy. The complexes and cells are examined and thecells are characterized. Examples of binding substances or reactivesubstances that may be used to coat the bead surface include, but arenot limited to: antibodies to specific cell surface proteins, smallmolecules that bind receptors or other cell surface molecules such asIL-2 or GM-CSF, avidin, biotin, or beads may remain uncoated insuspension that can interact by other means with cells. The antibodiesthat can be used are those known in the art. Many such antibodies areavailable from commercial companies, such as Zymed Inc., South SanFrancisco, Calif. and Dako Corp., Carpinteria, Calif.

Beads or microspheres can be made from a variety of substances includinggold, ferritin, polyacrylamide, or polystyrene. The latter is among thepreferred substances as beads can be made precisely to any sizespecification and can be uniformly conjugated to both molecular linkerarms and reactive binding substances. Polystyrene microspheres (alsoknown as “latex microspheres”) may be prepared by methods known in theart which are incorporated by reference herein. Binding substances thatcan be used include monoclonal antibodies, polyclonal antibodies,antibody “cocktail” mixtures, antibody fragments (such as Fc portions orFab or Fab′ fragments in either monovalent or divalent forms), smallmolecules that bind specific cell surface receptors, covalent andnon-covalent linkers, and indirect adherence such as utilizingelectrostatic or magnetic or paramagnetic attraction.

The prior art includes U.S. Pat. Nos. 5,554,505; 5,348,859; 5,340,719;5,231,005; 5,260,192; 5,338,689; 5,256,532; and 5,501,949, the entirecontents of which are incorporated herein by reference. These patentsinclude discussions of using certain microspheres or beads foridentification of cells. It is known in the art how to provide asuspension of cells in a liquid medium for analysis.

A second feature of a preferred embodiment of the present invention isthat it concentrates cells by using an appropriate filter without addedmanipulation of the cell suspension by cell lysis or added incubationsteps of submicroscopic paramagnetic microspheres. The filters to beused in the invention can be any of those known in the art, such asgynecological filters from Cytec Corp., Boxborough, Mass. The filterspreferably have a pore size larger than the beads being used so that allor most or substantial amounts of unbound beads and unbound cells passthrough, but the pore size is preferably small enough so that all ormost or substantial amounts of beads bound to cells are trapped on thefilter, such as the filter pore size being about 1, 2, 3, 4, 5, 6, 8, 10or 12 microns larger than the bead size. Preferred filter pore sizesinclude 10-15, less preferably 7-20, 7-30 or 7-40 micron pore sizes.Alternatively the filter pore sizes can be at least 15 or 20 microns.The filter is preferably mounted on a solid support, such as at the endof a tube through which the suspension can drain.

Other filtration methods can also be used. These filters include anysolid support that preferentially allows cell-bead complexes orindividual cells or beads below a given size to pass through the filterwhile those above a given size remain trapped in the filter. Otherfilter types that can be used include columns of solid materials such assepharose which contain a well-defined average pore size. Such columnsare known in the art. Another technique which can be used is to fixcoated beads using techniques known in the art to any solid support suchas a glass slide or a plastic substrate, such as an opaque plasticsubstrate. The cell suspension is then incubated on the slide (orsubstrate) or the slide (or substrate) is incubated in the cellsuspension. In this way the beads are contacted with the suspension. Thetarget cells adhere to the beads. Optionally a second and third or morebead type can be affixed to the cells as described herein to make largerbead-cell complexes. The suspension is then drained off the glass slideor substrate. In this way the bead-cell complexes are separated from thesuspension. If a glass slide was used, visualization can then be done.If some other substrate was used (for example, opaque plastic) the beadscan then be released from the substrate or support and the bead-cellcomplexes can be affixed to a glass slide for examination.

A cell suspension is preferably prepared from a peripheral blood sample,a bone marrow aspirate, a fine needle aspirate, a lymph node biopsy, ora body site specimen. In the method described herein, single parameter,simultaneous single parameter, and true multiparameter analysis ispossible which compares to the level of sophistication of analysispossible by flow cytometry. Beads that can be easily distinguished fromeach other optically either by size, color, or both can be added to acell suspension either simultaneously or sequentially. Positive bindingby the target cell population results in a bead-cell complex that has asignificantly larger physical size than either unbound cells or beads.These complexes can be then easily concentrated and separated from therest of the solution using an appropriately sized filter containingpores of sufficient size to let unbound cells and beads to pass throughwhile complexes remain on the filter. The method may also be used inreverse, in that abnormal cell populations may fail to bind beads whilenormal cells bind strongly. An example of this latter method can befound with the myelodysplastic disorders (MDS), which currently cannotbe diagnosed by flow cytometry with any degree of reliability. Normalhuman myeloid cells strongly express surface markers such as CD11b,CD13, CD15, CD16 and CD33. However, in MDS, these cell populations loseexpression of these markers. However, as normal cells degenerate fromprolonged storage or poor specimen handling such as temperatureextremes, which may occur in specimen transport, they also loseexpression of these markers. Flow cytometry cannot distinguish betweenthese two conditions. However, degenerated cells are easily recognizedmorphologically from the dysplastic cells of MDS. Loss of binding bybeads coated with antibodies to these markers could easily be identified(with a slide made of the cells passing through the filter as well asthose trapped on the filter). Therefore, this method may be used todiagnose MDS, heretofore only diagnosable in those minority of casesshowing abnormal cytogenetics or persistent hematologic abnormalitiesafter prolonged clinical follow up. As an example of MDS analysis, onecan look at a peripheral smear. If the cells are degenerated, get a newsample. If the cells are not degenerated, incubate the cell suspensionwith large beads coated with anti-CD13. Then add small beads coated withanti-CD15 and let react. Then filter (can be small pore size to trapboth bound and unbound cells, or large pore size to trap bound cellsonly, in which case unbound cells are collected from what went throughthe filter). If the result is many complexes such as those depicted inFIG. 3 and few unbound cells, this indicates normal cells. If there arefew bound cells and many unbound cells, this suggests MDS. Immaturecells also have weak binding, but this can be seen morphologically. Thesame procedure can be done with CD11b and CD16.

One can also count beads trapped on the filter prior to transfer to theglass slide. Using methods such as light scattering, reflectance,fluorescence, or electrostatic field changes, the number of beadstrapped on the filter can be counted. An average number of cells boundto each bead can be obtained and an estimate of the number of cells inthe original sample volume obtained.

With reference to FIG. 1 there is shown, not to scale, a bead 2, such asa polystyrene microsphere, which has coated thereon and bound thereto abinding substance 4 such as an antibody. There is also a cell 6, such asa target cell, which has a cell surface marker 8. The binding substance4 or antibody binds to the cell surface marker 8 on the target cell 6.FIG. 2 illustrates how this kind of reaction may appear on a glassslide; a group of cells or target cells 12 have bound to a bead 10. Thisshows single parameter binding of cells to beads. The ratio of beads tocells should be adjusted properly for effective results. The actualnumber of cells binding the bead is variable, ranging from a single cellto numerous cells crowding the bead's surface.

With reference to FIG. 3 there is shown a large bead 14 coated with abinding substance which has bound to eight cells 16, 18, 20, 22. Smallbeads or different colored beads 24 coated with a different bindingsubstance have bound to the cells 22 but not to the cells 16, 18, 20.This provides positive identification of target cells 22. Thisillustrates multiparameter analysis. Cells 22 is a subset of cells 16,18, 20, 22. A variable number of beads 24 can bind to each cell 22. Insome cases each cell bound to bead 14 will be bound to one or more beads24, or each cell bound to bead 14 may be unbound to small beads. Notethat different kinds of cells may bind to the large bead 14 that can insome cases be distinguished morphologically. Preferably the large bead14 is added first to the cell suspension so that a plurality of cellscan bind to its surface. Then the small beads 24 are added to bind tothe periphery of the complex. Alternatively small beads 24 can be addedfirst or small beads 24 and large beads 14 can be added simultaneously.The order of addition is dependent in large part upon the relativeconcentrations and surface areas of the beads and the cells. Forexample, you would not want to add beads 14 or 24 in such concentrationsthat they completely cover or obscure the surface area of the targetcells and thus prevent access thereto by the other beads. Preferablythere is an excess of target cells to fully coat the bead. Optionallythe suspension can be filtered after the first complex is formed, totrap the first complex and resuspend it before the second beads areadded. Thus a group of complexes can be filtered and resuspended beforea subsequent set of beads is added; this can lead to more certain anddistinct results by removing materials which would provide interference.The beads may be distinguishable in size or color or both. Furtherlevels of multiparameter analysis can also be carried out, such as byadding to FIG. 3 another set of different sized or different coloredbeads which would bind to a first subset of cells 22 but not theremaining cells 22, thus providing positive identification of said firstsubset of cells 22. In this manner subsequent or additional levels ofmultiparameter analysis can be carried out.

There is a wide variety of available beads that can be used, and thoseselected would depend on the specific application. In multiparameteranalysis beads that can be easily distinguished by either size or colorare preferable. For example, two sets of colorless beads sized 10 and 5microns respectively can be used to isolate a population of B cellsusing 10 micron beads coated with an anti-pan B cell antigen such asCD19 and 5 micron beads coated with anti-kappa. Multiparameter analysisthat cannot be easily mimicked by flow cytometry is available by a minorvariation of this example. Colorless 10 micron beads are used to bind Bcells by using anti-CD19 coated beads. 5-micron colorless beads arecoated with anti-kappa while dark blue 5-micron beads are coated withanti-lambda. Similarly, a blast cell population can be analyzed usinganti-CD34 coated 10 micron beads and anti-CD19 coated colorless 5 micronbeads. Colored 5-micron beads coated with anti-CD13 are simultaneouslyadded for rapid characterization of most blast cell populations.

Preferred Methods:

1) Substantially identical beads are purchased commercially precoatedwith strepavidin (Bangs Laboratories, Fishers, Ind.). A small quantityis suspended in any buffered salt solution such as phosphate bufferedsaline or commercially available antibody diluent. The beads areincubated with biotinylated goat anti-mouse antibodies for 30 minutes(however, any biotinylated anti-allogeneic antibody may be used). Thesuspension is centrifuged and the supernatant drawn off. The incubationis repeated two times to ensure coating of as much of the availablesurface area of the beads as possible. The beads are then washed threetimes using the same buffer. The suspension is then incubated withspecific mouse anti-human antibodies for 1 hour (or any non-biotinylatedanti-allogeneic antibody specific for the target cell population may beused). The suspension is again washed three times and diluted to thedesired concentration. The resulting suspension can be refrigerated at 4degrees Centigrade until use. Alternatively, biotinylated primaryantibodies may be used without the use of secondary antibodies. Thebeads produced by this technique are substantially identical.

2) Beads are precoated with anti-Fc receptor antibodies (BangsLaboratories, Fishers, Ind.) such as goat anti-mouse IgG Fc receptorantibodies. These beads can then be suspended in a solution ofantibodies which would spontaneously bind to the anti-Fc receptor siteson the beads. In the example cited above, mouse anti-human antibodieswould be bound to the beads followed by appropriate washing stepssimilar to that described above.

3) Binding substances such as any protein, peptide, or nucleotidesequence may be bound by other chemical or specific binding methods. Forexample, polystyrene microspheres are “naturally” left coated withsulfate surface groups after manufacture. These ligands can be used tolink proteins and peptides directly to the surface of the beads.Examples of such functional surface groups that can be coated on thesurface includes, but is not limited to, aldehyde, aliphatic amine,amide, aromatic amine, carboxylic acid, chloromethyl, epoxy, hydrazide,hydroxyl, sulfonate, and tosy (toluene sulfonyl) reactive ligands. Thesecan then be used in turn to link peptides, proteins, oligonucleotides,and other biochemical ligands to the surface. These ligands or bindingsubstances would in turn be used to bind specific sites on cell surfaceswhich would link the cell to the surface of the bead. For example, asmall molecule such as the hormone IL-2 could be used by one of theabove methods to coat beads with the intention of binding IL-2 receptorsites (CD25) on cell surfaces. This could be used to bind cells such asT-cells, monocytes, and neoplastic cells such as hairy cell leukemia.

Other Methods:

Submicroscopic paramagnetic microspheres (preferably less than 1 micronin diameter) are bound to any reactive biomarker of interest. Thebinding that is used could be any of the above methods. Cells are thenpermeabilized and fixed using a variety of detergents and weak fixativesolutions such as 1% paraformaldehyde. Alternatively a number ofcommercially available permeabilizing kits are available for thispurpose such as IntraStain (Dako Corp., Carpinteria, Calif.). Thereactive biomarker, such as antimyeloperoxidase antibodies,anti-terminal deoxytidyl transferase antibodies, or specific RNA or DNAprobes, is then incubated with the cell suspension. The biomarkers andparamagnetic particles get inside the cell and, for example, the probebinds to the intracellular target. The cells are then washed andresuspended in a suitable buffer such as PBS or RPMI. The suspension isthen incubated with magnetic beads or microspheres of a size or coloreasily visualized, such as 1 to 20 or 3-15 or 5-10 or 10-20 microns. Themagnetic beads bind to the cell surface, but cannot cross the membrane,to create a cell-bead complex that is easily trapped such as viafiltration.

In one example, abnormal blasts in a bone marrow suspension can bepermeabilized and incubated with anti-myeloperoxidase antibodies boundto submicroscopic paramagnetic microspheres. The suspension is thenwashed three times in buffered salt solution and resuspended andincubated with large magnetic beads of a preferred size of 5-15 microndiameter to create cells bound to large beads.

In another example, specific DNA sequences (probes) are bound tosubmicroscopic paramagnetic microspheres using methods such asavidinated microspheres and biotinylated probes. Cells from a patientwith chronic myelogenous leukemia are permeabilized and incubated withprobes binding to the specific bcr-abl translocation that is diagnosticfor the disease. The suspension is then washed and incubated with largemagnetic beads of a preferred size of 5-15 micron diameter to createcells bound to large beads.

Detection and Analysis:

The cell-bead complexes (cells bound to beads) provided or obtained asdescribed above are then passed through a solid support filter having aporosity of sufficient size to allow unbound cells and beads to passthrough. There are several acceptable filtration techniques such asporous columns and solid supports. In a preferred method, the suspensionis passed through the filter using a variety of acceptable methods whichincludes gravity, suction (applied vacuum), positive pressure on thefluid side, or wicking the fluid through the filter using a porousabsorbable material such as gauze pads. Various devices that can be usedinclude pistons, syringes, or suction methods to create a negativepressure to pass fluid through the filter. In a preferred embodiment, asingle solid filter with a pore size of 10-15 microns is used. Cell-beadcomplexes remain trapped on the filter and the layer is then transferredto a glass slide by direct contact with the slide and applying gentlepressure. The resulting slide preparation can be stained using a varietyof commercially available stains such as hematoxylin and eosin,Papanicolau stain, or any Romanowsky stain. In a preferred embodiment,the cells remain suspended in a compatible buffer such as PBS, RPMI, orcommercially available antibody diluent and the resulting slide isstained with Wright-Giemsa stain. Alternatively, cells may be suspendedin ethanol or a commercially available fixative such as Cytolyte (CytycCorp., Boxborough, Mass.). The resulting slide is then stained withPapanicolau or hematoxylin and eosin stains. The complexes are examinedand the cells are characterized under routine light microscopy.

The invention can be used to perform single parameter analysiscorrelated with morphology, simultaneous single parameter analysis, ormultiparameter analysis. In single parameter analysis, (depicted inFIGS. 1 and 2) a single bead type is added to a suspension of cells in aliquid medium so that after filtration the slide is provided with anenriched single cell population. This is useful as a simple screen todetermine if a cell population has a particular characteristic such asdistinguishing monocytes from monocytoid B lymphocytes as cited inExample 1 below. In this configuration, cells bind to beads and arevisible on the glass slide for analysis. Alternatively, a B cellpopulation can be assayed for expression of kappa or lambda by using twoseparate slides or slide wells each of which contain a single bead type(anti-kappa or anti-lambda). Another variant of this analysis is to addsimultaneously to the cell suspension two different bead types, oneanti-kappa and a second anti-lambda. This is an example of simultaneoussingle parameter analysis since binding of each bead type is independentof the other but the results are analyzed together. An analogoussituation occurs in flow cytometry analysis when fluorescence isdisplayed vs. cell number to obtain a single histogram. In kappa andlambda analysis, a monoclonal population can only be detected bysimultaneous analysis of both histograms and looking for single peaks offluorescence. Finally, multiparameter analysis can be performed bylinking detection of two different characteristics so that analysis isperformed together. In this case, binding of one set of beads occurs,followed by a second and optionally more sets of beads (see FIG. 3).Analysis looks for simultaneous binding of more than one set of beads tothe target cell population (as depicted in Example 2 below).

The invention can be used to detect abnormal loss of binding when strongbinding would be expected. For example, normal myeloid cells such asmature granulocytes and monocytes in the peripheral blood would beexpected to strongly express the surface markers CD13, CD33, CD11b, andCD16. In a bone marrow sample there would be a continues range ofincreasing expression of these markers as the cell matures. However,cells showing abnormal maturation, as seen in myelodysplasia, would showdiminished expression of these markers. This phenomenon has beenpreviously described by Davis, et al. and can be seen in flow cytometryanalysis as abnormal patterns of expression on appropriate histograms.However, a similar loss of expression is seen when normal cells die anddegenerate as occurs in specimen mishandling or aging. Since morphologiccorrelation is less than optimal by flow cytometry, the phenomenon haslimited diagnostic usefulness, particularly when the specimen has beentransported long distances. In the present invention, cells can bevisualized on the glass slide to confirm their viability. Normal cellswould strongly bind beads coated with these markers but there would bedecreased binding of beads in cells with myelodysplasia. In the lowgrade myelodysplasias such as refractory anemia and refractory anemiawith ringed sideroblasts, there are often no objective diagnosticcriteria for confirming the diagnosis. Current state of the art in suchcases requires prolonged follow up and diagnosis by exclusion of otherpossible entities such as ethanol toxicity or megaloblastic anemia fromvitamin B12 or folate deficiency. The invented method provides a muchneeded positive diagnostic test.

A complementary detection method is that prior to transfer of the cellsto a glass slide, the filter is gently rinsed and scanned using a lightbeam of either a white light beam or a specific wavelength to correspondto the excitation wavelength of fluorescent beads. The number of eventsis counted electronically and the cells are then transferred to a glassslide and stained. The average number of cells per microsphere is thenobtained manually and an estimate of the total number of target cells inthe sample can be estimated (assuming that a known volume of sample isused).

Preferred Applications:

1) Single parameter analysis of tumors and other specific cellpopulations. A suspected tumor with a known immunophenotype can beanalyzed to confirm the presence of a single marker as outlined inExamples 1 and 3 below. This is most useful in settings where a singleissue regarding cell phenotype needs to be settled. In Example 1 below,knowing that the abnormal cell population is of B cell origin issufficient information to proceed with further studies, since thissuggests (but does not prove) malignancy. In Example 3 below, knowingthat the lymphoid population is of T cell origin suggests that thepatient has a reactive infiltrate rather than a malignant infiltrate. Ifthis assay had been clinically available in both of these unusual cases,the results of the simple study in Example 1 would justify furtherexpense of additional evaluation. The results of Example 3 justify notperforming flow cytometry and proceeding to treatment for meningitis.Other applications of these kinds of analysis can be useful in otherkinds of tumors such as MN/CA9 screening for cervical cancer,identifying specific tumor types in malignant infiltrates such asmelanoma (using markers such as HMB-45), or identifying micrometasticdisease in lymph nodes and bone marrows. In addition, single parameteranalysis can be used in genetic phenotypic and genotypic analysis. Forexample, a peripheral blood sample can be permeabilized and treated witha specific probe to the bcr-abl translocation. The probe can be labeledwith paramagnetic submicroscopic microspheres. The cells can then betreated with large, magnetic beads to identify the presence of thetranslocation that would be diagnostic of chronic myelogenous leukemia.Alternatively, a similar method can be used to identify the presence ofintracellular proteins or RNA sequences using appropriate antibodies ornucleotide sequences, for example, the expression of the intracellularprotein terminal deoxyribonucleotidyl transferase (TdT) using anantibody also labeled with paramagnetic microspheres and detecting thereaction using large surface magnetic beads. Finally, the use of CD64expression has been proposed as a rapid diagnostic test for clinicallysignificant acute inflammatory reaction (Lab. Hematol. 1995; 1:3-12).For reasons described above, flow cytometry is too expensive anddifficult to use as a screening procedure for common conditions. Theinvented method allows rapid, inexpensive single parameter analysis forCD64 expression in peripheral granulocytes.

2) Simultaneous single parameter analysis is where there is simultaneousanalysis of markers that are independent of each other. Most commonly,this is used in a B cell lymphoid population to determine expression ofeither kappa or lambda light chain restriction by expressed surfaceimmunoglobulins. This can either be done by using similar beads as usedin two separate glass slides analyzed simultaneously or by using asingle slide using two sets of beads which can be easily distinguishedbased on size, color, or both. This is extremely useful as aninexpensive, rapid screen for B cell monoclonality. Other useful typesof simultaneous single parameter analysis are in the setting of amalignant tumor of unknown origin where a cell suspension can beanalyzed, either by using multiple separate slides or a single slidecontaining multiple sets of beads that can be distinguished by size,color, or both. In this example, these sets of beads typically includebeads marking for CD45 (leukocyte common antigen), HMB-45 (melanoma),and a general cytokeratin marker (often AE1 and AE3 cocktail forepithelial tumors). A third type of this kind of analysis is to screen apopulation of lymphocytes to determine whether this population iscomposed of B cells, T cells, other cells, or any combination of thesetypes.

3) The invention also includes multiparameter analysis where expressionof markers are analyzed in conjunction with other markers. A simple, butcommon, example of this kind of analysis is depicted in Example 2 below.In Example 2, the positive binding reaction by the anti-CD20 coatedbeads which isolates the B cells is linked to kappa or lambda lightchain expression. Multiparameter analysis enhances analysis sincecorrectly identifying certain cell populations requires logicalassociation of multiple subsets of markers. A case of acute leukemiaserves as a useful example of this kind of analysis. Morphologicexamination is one of the best methods for identifying the abnormalblast cells, but it does not characterize the kind of blasts present.Combining morphologic analysis with the present invention would yieldthe following typical kind of analysis. Anti-CD34 coated beads arecombined with anti-HLA-DR coated beads to confirm expression of both ofthese markers in the malignant cell population. Positive expression ofboth of these markers supports the diagnosis of acute leukemia. Thecells can then be analyzed with anti-CD13 and anti-CD33 coated beads inconjunction with anti-CD19 and anti-CD2 coated beads to determine if thecells are myeloid or lymphoid in origin. If they bind to CD13, CD33, orboth, this confirms the myeloid derivation of the cells. The cells canalso be analyzed with anti-CD15, anti-CD14, anti-CD56, anti-CD7, andanti-CD4 to determine subtype (myeloid, monocytic, or both) and to yieldprognostic information. Of particular interest is successful analysis ofacute promyelocytic leukemia (FAB subtype M3). Analysis of this tumortype by flow cytometry is fraught with errors and the tumor can bemissed since it is composed of maturing myeloid cells. Using the presentinvention, morphologic analysis would confirm the presence of excessnumbers of promyelocytic cells. In addition, the promyelocytes wouldusually be HLA-DR negative and could also be analyzed for thetranslocation of chromosomes 15 and 17 (t(15;17)) which is diagnostic ofthe disease. This kind of analysis is particularly useful in themicrogranular variant of the disease in which the cells may resemblemonoblasts. Monocytic leukemias can also be analyzed for additionalmonocytic markers such as CD36. Similar kinds of analyses can beperformed for other hematologic malignancies, other tumor types, andother specific cell populations. In addition, the method can be used inreverse to offer a diagnostic test for myelodysplasia. Normal myeloidcells strongly bind the myeloid markers CD11b, CD13, CD16, and CD33.Among the changes seen in myelodysplasia, is decreased expression ofthese markers by flow cytometry. However, degenerating cells, as occursin excessive sample age, temperature extremes, or other forms ofspecimen mishandling also causes decreased expression of these markers.Since morphologic correlation with flow cytometry is so poor, this formof analysis has not gained significant clinical acceptance since flowcytometry cannot reliably distinguish between degenerated normal cellsand myelodysplastic cells. In the invented method there is excellentmorphologic correlation, and trained observers will easily recognizedegenerated cells. Therefore, normal cells can easily be distinguishedfrom dysplastic cells, as normal cells will avidly bind beads coatedwith antibodies to these markers and dysplastic cells will not.

Another similar application can be used for analysis of breast cancer todetermine prognostic factors such as Her2/neu overexpression. Currentstate of the art utilizes primarily immunohistochemistry to localizeactual tumor from surrounding breast tissue by visual methods. Her2/neucytoplasmic membrane expression is estimated by the observer visually ona scale expressed as 0+ positive (no expression) to 4+ positive(strongest possible expression). There are semi-automated methods usingimage analysis that objectively measure Her2/neu staining but these areexpensive and have limited availability. Alternatively, Her2/neuexpression can be more objectively estimated by using fluorescentin-situ hybridization (FISH) which labels each gene copy with afluorescent dot. The number of gene copies in each cell can be estimatedby merely counting the dots within the nucleus of each cell. However,because cells cannot be easily counter-stained and observed, it isdifficult to tell a malignant breast epithelial cell from an admixedbenign one or even a stromal cell from the breast supporting matrix.Therefore, analysis by FISH has less acceptance in the clinical setting.More recently, Her2/neu expression can be performed by flow cytometry,however, like FISH there is no method for evaluating whether theanalyzed cell is a malignant cell or a benign one. Using multiparameteranalysis as described in the present invention, epithelial cells in acell suspension can be distinguished from stromal cells by using large(10 micron) beads coated with anti-cytokeratin antibodies. Onlyepithelial cells would bind to this bead. Small 5 micron beads coatedwith an appropriate anti-Her2/neu antibody is then added to the mixtureand the suspension filtered. Her2/neu expression can be analyzedobjectively by several methods. In one method, the filter itself can beanalyzed to determine the quantity of 5 micron beads present on thefilter by using methods such as fluorescence (if the 5 micron beads arefluorescent), electrostatic assessment, or other of a variety of knowncounting methods. In an alternative method, the suspension istransferred to a glass slide after filtration and the slide stained.Benign cells can be distinguished from malignant ones by morphologicassessment and the average number of beads binding to malignant cellscan be estimated. This can either be performed manually by the observeror in a semi-automated manner using an electronic visual analysis tocount the number of beads bound to each cell identified by the observeras malignant.

4) Signal amplification of weakly expressed antigens. One of the majoradvantages of flow cytometry is its ability to detect weakly expressedantigens on the surface of cells. Many antigens fall under this categoryand cannot be easily detected using alternative means such as routineimmunostains using standard colorimetric detection methods such asdiaminobenzadine (DAB). This problem in immunostains has been partiallyovercome using signal amplification methods such as tyramide signalamplification which is commercially available such as the CatalyzedSignal Amplification kit (Dako Corp., Carpinteria, Calif.). In themethod, the primary antibody is conjugated to peroxidase enzyme (usuallyhorseradish peroxidase or HRP) and oxygen free radicals are generated.In the presence of tyramide, the tyramide molecules themselves becomefree radicals and are short lived, highly reactive species. They readilyconjugate to nearby molecules and are fixed in the immediate area of theprimary antibody. The signal amplification derives from the ease inwhich tyramide is conjugate either to a fluorescent molecule orperoxidase. This added peroxidase is used to generate additional DABsignal and thus the signal is amplified. This signal amplificationtechnique can also be applied to the invented method described herein.In one example, primary antibodies are conjugated to HRP to generatebiotinylated tryamide free radicals as per the manufacturer'sdirections. Avidinated beads then readily and spontaneously bind to thecell surface at the appropriate sites. An alternative method usessubmicroscopic beads that are invisible by routine light microscopywhich are coated with the antibody of interest that also have a peroxidefree radical generator such as HRP bound either to the antibody or tothe surface of the bead. Biotinylated tyramide free radicals aregenerated as per the manufacturer's directions and then the cells arewashed (or filtered) and treated with avidinated large beads that areeasily visible by light microscopy (typically beads in the 5-20 micronsize range). This method of signal amplification greatly enhancesotherwise weak binding of beads when only rare antigens are present onthe cell surface. Single amplification can also be achieved using (1)the dual-labelled Envision polymer system available from Dako Corp.,Carpinteria, Calif. or (2) the mirror image complimentary antibodiestechnique, a kit for which is available from The Binding Site Company,Birmingham, England.

5) An alternative method of multiparameter analysis can be performed byfirst using a single set of beads to isolate the target cell population.The second parameter can then be detected by using routine orconventional immunohistochemical techniques such as immunoflouresence,colorometric methods such as peroxide reduced DAB or alkalinephosphatase methods, or immunogold/silver enhancement. This secondantibody detection system can be applied either in the cell suspensionor after the slide is made but before it is stained. The choice ofmethod and detection method would be dependent on the desired stain inthe final product and the particular antibody to be used. Since thismethod bypasses fixation and processing used in paraffin embedded tissuesections, antibodies that cannot be used in these paraffin can be usedhere such as CD10, CD2, or CD19.

6) Cell-bead binding is potentiated by the use of one or moreenhancement reagents. In a preferred application, albumin, preferably22% bovine albumin (Ortho Diagnostics, Raritan, N.J.) is added as anenhancement reagent to the cell suspension prior to the addition ofbeads. Albumin not only enhances reactions, it stabilizes cells andpreserves cells during the remainder of the procedure. Other enhancementreagents can less preferably be used, such as various molecular weightsof polyethylene glycol (PEG). PEG of a wide range of molecular weightsand subgroup attachments can be used, but PEG 200 through PEG 3350 (PEGwith molecular weights of 200 kD through 3350 kD) are preferred. Otherantigen-antibody enhancement reagents such as low ionic strength saltsolutions may also be used. When albumin or PEG solutions are used, anamount equal to approximately 5-20% w/v should be added to the cellsuspension.

7) In order to reduce the possibility of granulocytes in cellsuspensions from recognizing beads as foreign bodies and phagocytozingbeads, a very low concentration (1 or 2 drops/ml) of any of a variety offixatives may be added to the cell suspension. The preferred fixativefor this application is ethanol, but formaldehyde, paraformaldehyde,methanol or glutaraldehyde are among the many commercially availablefixatives that can be used for this purpose. If such fixatives areadded, the cell suspension should be washed using a suitable cellsuspension medium prior to adding beads. Failure to do so will inhibitbinding of beads to cells. An alternate method of inhibiting thisnon-specific phagocytosis is a specific inhibitor of this particularcell function. These compounds act without causing cell death and may beuseful in specific settings where preservation of cellular activity isdesired. An example of this class of reagent is colchicine, whichinhibits cytoplasmic microtubule formation. This inhibition blocksphagocytosis.

8) In situations where cell analysis cannot be performed within severalhours of obtaining the sample, the cell suspension can be easilypreserved for later analysis. The sample is simply added toapproximately an equal volume of fixative, preferably a non-crosslinkingfixative such as methanol or ethanol. A suitable commercially availablefixative for such a purpose is Cytolite (Cytyc Corp., Boxborough,Mass.). Cross-linking fixatives such as formaldehyde or glutaraldehydemay also be used but this will limit the range of available antigensthat can be analyzed. Cells preserved in this manner may be refrigeratedfor up to 10 days. In order to analyze the sample, it must be washedthree times in a suitable buffer such as PBS with albumin. Analysis ofthe sample may then proceed as described above.

9) A specific cell type in a cell suspension may be quantitated usingthe following technique:

a) An absolute cell count per unit volume is obtained by an independentmethod. This can be done manually using a hemocytometer but acommercially available cell counter is the preferred method.

b) A specific quantity of the cell suspension, for example 1 ml, is usedfor the reaction. Therefore, a known total number of cells is used inthe analysis.

c) Beads coated with specific binding agents are added to the suspensionand allowed to react, i.e., bind to cells of interest to form complexes.

d) Filter out the complexes and place them on a glass slide and stainthem.

e) Relative percentages of each cell type is then estimated by countingthe number of cells on each specific bead type.

f) These percentages are used to calculate absolute numbers of cellpopulations per unit volume in the original cell suspension.

Using this approach, at least two different kinds of analysis can beaccomplished. First, an analysis can be done where the total cell countequals A and cell count B plus cell count C approximately equals cellcount A. Second, an analysis can be done where the total cell countequals A and there is a subset of A which is cell count D.

EXAMPLE

The physician needs an estimation of the absolute count of the CD4+lymphocytes in a peripheral blood sample. The peripheral blood sample isanalyzed with a hematology cell counter to obtain an absolute count oflymphocytes/ml in the peripheral blood. Four different bead populationsare coated with anti-CD20, anti-CD3, anti-CD4, and anti-CD8 antibodies,respectively, and are added to the suspension. These four different beadtypes or sets can be distinguished from each other by size, color, orboth. For convenience, these sets of beads are preferably split into twoseparate analyses: a first tube for CD20 and CD3 positive cells and asecond tube containing large CD3 beads and small CD4 and CD8 beads. Tothe first tube, two different bead sets are added, one for CD20 andanother for CD3. A specific quantity of peripheral blood sample such as0.5 ml is added to the tube. Incubation and filtration (or separation)as described above is performed and one or more slides are prepared.From the first slide containing CD20 and CD3 cells, the relativepercentages of B versus T cells can be estimated because the beads are adifferent color or a different size. From these percentages the absolutenumbers of these cells per unit volume in the original sample can becalculated, and the two added together should almost equal the totalabsolute lymphocyte count (this can serve as a control). To the secondtube, three different types of beads are added, a large bead for CD3, afirst small bead for CD4 and a different small bead for CD8. Processingproceeds as described above. From the second tube the relativepercentages of CD4+ lymphocytes versus CD8+ lymphocytes can becalculated and the absolute number of each obtained. This methodeliminates the problems of interfering monocytes (which are also CD4positive) which has to be corrected for in flow cytometry since usingthe proper filter size, they would not be present on the final slide.Even if they are on the final slide, they can be ignored because the CD3beads are not bound to them.

Alternatively, an absolute count may show 200 Type A cells per unitvolume. Beads Y and Z are added. Bead Y binds all Type A cells; bead Zbinds Type D cells, which is a subset of Type A cells. After separatingthe complexes, examination shows one Type D cell for every ten Type Acells. This then calculates to an absolute count of 20 Type D cells perunit volume.

In the various cases mentioned above, a second or smaller or differentbead is added to a tube or suspension to act as a marker. Optionally,instead of a second or smaller or different bead, one can instead use anon-bead marker such as a DNA or RNA probe, an immunohistochemicalmarker or a cytochemical stain. For example it is possible to quantitatethe number of malignant cells in a case of follicular lymphoma in aperipheral blood or bone marrow sample. In this example, the number oflymphocytes is quantitated (in this example, 200 lymphocytes per ml)using an automated hematology analyzer. CD20 positive fluorescent beadsand non-fluorescent CD3 beads are added to the suspension that aredistinguishable from each other by size as well. Under routine lightmicroscopy, the relative percentages of B cells and T cells isdetermined, for example, 25% B cells and 75% T cells. The absolutenumber of B-cells is then calculated by multiplying the relativepercentage of B-cells (25%) by the absolute number of lymphocytes (200per ml) to yield 50 B cells per ml. The slide is then treated byfluorescent in-situ hybridization for the IgH-bcl-2 gene rearrangement(t(14;18)) that is a marker of follicular lymphoma. This method isreadily available from several commercial manufacturers such as Vysis,Inc. (Downers Grove, Ill.). Using a fluorescent microscope, the numberof B-cells (as identified by the fluorescent bead) that co-expresses thein-situ fluorescent signal is counted and the relative percentage vs.the negative signal cells is determined. For example, of the B cells, itis noted that 1 in 5 (ie, 20%) co-express the in-situ fluorescentsignal. Therefore, it is calculated that 20% of the 50 B cells per ml ismalignant, ie, 10 follicular lymphoma cells per ml. Many variations ofthis method are possible using routine histochemical, cytochemical,immunohistochemical, and fluorescent markers.

10) The invention may also be used to prepare a cell suspension (ratherthan glass slides) that is enriched for the target cell suspension. Inthis variation, rather than transferring the cell-bead complexes to aslide for visual or other analysis, the filter may be gently washed toresuspend the cell-bead complexes. The filter (or variations describedherein) may be rinsed either by reversing the fluid flow through thefilter using clean buffer or by simply rinsing the filter in a suitablebuffer. The cell-bead complexes can then be used for other purposes orreanalyzed by other methods. If the appropriate linker molecules asknown in the art are used to attach the antibodies to beads, theselinkers may be broken using chemical methods as known in the art such asenzymes. This will release the cells from the beads and make themavailable for other studies such as tissue cultures. Optionally one mayuse these cells for clinical uses as well. For example, a bone marrow orperipheral blood sample may be treated with anti-CD34 beads in which theanti-CD34 antibodies are bound to the beads using a reversible orcleavable bond. The CD34+ stem cells are then trapped on the filter andthe filter is then washed using a suitable buffer. The stem cells arethen released from the beads by cleaving the bonds holding the anti-CD34antibody to the bead. The cells can then be separated from the beadsagain by filtration using appropriately sized beads and filter pores.For example, the beads can be 10 micron in diameter and a filter set at7 microns can be used. This will trap the now unbound beads on thefilter and the stem cells will pass through the filter.

The following Examples further illustrate various aspects of theinvention, including single parameter and multiparameter analysis.

Example 1

A 30 year old man presented with pancytopenia and splenomegaly.Examination of the peripheral smear confirmed the pancytopenia. Inaddition, scattered cells were present that showed bland cytologicalcharacteristics, with a monocytoid appearance. The nuclei of these cellswere round to oval, with a single intermediate nucleolus. There wasabundant blue-gray cytoplasm that showed numerous cytoplasmicprojections. A bone marrow examination revealed a hypocellular aspiratewith similar cells present. Small clusters of abnormal cells werepresent on the core biopsy. A buffy coat sample of the peripheral smearwas suspended in anti-CD20 coated 10-micron colorless beads todistinguish the abnormal cells from monocytes. The suspension was passedthrough an appropriate filter and the cells were then transferred to aglass slide and stained. A schematic of the resulting slide preparationis demonstrated in FIG. 2. Positive binding of the abnormal cellpopulation to the 10-micron beads was a suspicious finding and suggestedan abnormal B cell population. Flow cytometry performed on the bonemarrow aspirate revealed a monoclonal population of monocytoid B cellsexpressing CD19, CD11c, CD103, and kappa light chain restrictionconfirming the diagnosis of hairy cell leukemia.

Example 2

A 68 year old man with a known history of chronic lymphocytic leukemia(CLL) presented for routine follow up examination. Clinical examinationrevealed that the patient had a peripheral white cell count of 435,500cells/ml (normal range 4,300-11,000 cells/ml) which included 87%lymphocytes. Morphologic examination of the peripheral blood smearrevealed predominantly an abnormal population of small lymphocytes witha small but significant population of large transformed cells. Asuspension of cells in a liquid medium was provided. This sample wasanalyzed using anti-CD20 coated 10-micron beads, anti-kappa coatedcolorless 5-micron beads and anti-lambda coated colorless 5-micron beadsin two separate tubes. In the procedure, the same sample was placed intoeach of 2 tubes. To each tube was added anti-CD20 coated 10-micronbeads. These strongly bound the B cells. The question then was whetherthe B cells were kappa, lambda or a combination of both. Therefore, the5 micron anti-kappa beads were added to the first tube and the 5 micronanti-lambda beads were added to the second tube. The results were thenanalyzed after filtering and placing on a glass slide. The cellsstrongly bound to the 10-micron beads and showed no binding to theanti-lambda beads and scattered binding to the anti-kappa beads (ie,like FIG. 3, except only 1-2 small beads per complex). These results aretypical of CLL since this tumor strongly expresses CD20 but weak lightchain restriction when analyzed by flow cytometry. As an alternativeprocedure, the 5 micron anti-kappa beads could be red and the 5 micronanti-lambda beads could be blue. The procedure could still be in 2 tubesas described above, or the kappa and lambda beads could be addedsimultaneously to the first tube. Analysis of this latter result wouldshow a complex like FIG. 3 with blue only around the periphery(indicating monoclonal lambda), red only around the periphery(indicating monoclonal kappa), or a combination of red and blue aroundthe periphery (indicating polyclonal B cells).

Example 3

A 19 year old man presented with headache and stiff neck to theemergency. His evaluation included obtaining a sample of cerebral spinalfluid for which emergency pathologist evaluation of the fluid wasrequested to rule out the presence of “blasts”. Evaluation showed arelatively uniform population of small lymphocytes, and a diagnosis ofviral meningitis was suggested. The patient's physician requested flowcytometry to completely rule out the possibility of malignancy. Sinceexcess fluid was available, a small sample was treated with anti-CD20coated 10-micron beads and anti-kappa and anti-lambda coated 5-micronbeads in two separate tubes using essentially the same procedure asdescribed in Example 2 above. The majority of cells did not bind toeither the anti-CD20, anti-kappa, or anti-lambda beads, suggesting thatthe lymphoid population was composed predominantly of T cells. Flowcytometric analysis received two days later confirmed approximately 60%T cells and 40% B cells with normal T cell subsets and polytypic B cellsconsistent with viral meningitis.

A major advantage of the invention is that analysis of cell populationscan now be performed by simple inspection of the glass slide by anyphysician or technologist. This kind of analysis can be used on any typeof cell population bearing specific cell surface markers and in a widevariety of conditions (lymphoma is one example). Malignant clones frompatients with acute leukemia can be similarly analyzed (using differenttypes of markers), as can cell populations from patients with acquiredimmune deficiency syndrome. Finally, as tumor markers for solidneoplasms become available, this kind of analysis can also be performedin a similar fashion. For example, the new MN/CA9 antibody appears to bespecifically expressed by dysplastic and malignant uterine cervicalsquamous cells. Since these cells may be suspended in a sea of normalcells, they may be difficult to identify even by routineimmunohistochemistry. This method of analysis may both identify thesecells and enrich a cytological preparation for them so that they can bemore easily analyzed.

The present invention also provides a kit for practicing the invention.The kit contains one or more sets of beads as described above. Each setof beads is preferably in a container such as a sealed test tube. Insome cases of simultaneous single parameter or multiparameter analysis,two or more sets of beads can be premixed, but typically they are keptseparated. The kit also preferably contains one or more appropriatefilters as described above and preferably a set of instructions.

The methodology described herein can be automated and condensed. Anexample of a semiautomated device 25 for the performance of this kind ofanalysis is depicted in FIG. 4. A sample is prepared to make a cellsuspension. The sample is then loaded into the machine 25 in the sampleloader 26 and the machine 25 is programmed for the kind of analysisdesired (lymphoma screen, acute leukemia analysis, myelodysplasia,etc.). The sample is divided into the appropriate number of reactionchambers 28 (for example, 2, 4, 6, 8, 10 or 12) and a preprogrammednumber of bead sets (for example, 1, 2, 3, 4, etc. bead sets) addedsequentially or simultaneously to each reaction chamber. The beads areincubated in the cell suspension and allowed to bind to the cells andall reaction chamber samples are then transferred to a filtrationchamber 30 where each reaction chamber sample is filtered. The resultingfilters or filtered materials are arranged so that all of them aresimultaneously transferred to a single glass slide such as glass slide32. The resulting slide contains a series of wells, each wellcorresponding to a reaction chamber sample. The multi-well slide can bestained, then scanned under a microscope. Each well can correspond to amultiparameter analysis, which is performed in minutes. FIG. 5 is aschematic for a suggested lymphoma panel slide using such a procedure.FIG. 5 shows 6 wells, each having run a 3-bead set as shown formultiparameter analysis. In the upper left hand corner is“CD20/kappa/lambda”. This indicates a well where the machine ran theCD20/kappa/lambda analysis described earlier herein. The other 5 wellsgive antibody information for running similar analyses as known in theart. Optionally a fourth or fifth set of beads can be added for furtherlevels of analysis. Preferably after the single parameter ormultiparameter incubation and filtration is carried out, the resultingcomplexes (such as in FIG. 3) are stained by immunohistochemistry orin-situ hybridization and then evaluated. Coated glass slides arepreferred, to increase adhesiveness. Preferably, the slides are stained,coverslipped and examined by routine light microscopy to assess binding.Cells bound to beads are preferably assessed to characterize and ensurecell type.

In the present invention cells in suspension in fixative or tissue mediacan be phenotyped by antibody coated beads and isolated from thesurrounding milieu by the use of a filter of proper pore size. Thesebound cells, thus separated from the sea of other cells, can betransferred to a glass slide and stained with a variety of stains forvisualization. In addition, if immunophenotyping is not desired, aroutine cytologic preparation using a variety of methods such ascytospin, cell block, or ThinPrep can be prepared.

Single parameter analysis can be used to phenotype cells of interest,such as enumerating relative numbers of kappa and lambda-bearing Blymphocytes. Another application is the isolation of MN/CA9 positivecervical epithelial cells.

Certain cell surface markers can be semi-quantitated by first isolatingcells of interest and then enumerating the average number of beads boundto the surface.

It should be evident that this disclosure is by way of example and thatvarious changes may be made by adding, modifying or eliminating detailswithout departing from the fair scope of the teaching contained in thisdisclosure. The invention is therefore not limited to particular detailsof this disclosure except to the extent that the following claims arenecessarily so limited.

What is claimed is:
 1. A method of processing cells comprising the stepsof: a) providing a suspension of cells in a liquid medium, said cellsincluding first cells, b) contacting a group of first beads with saidsuspension, each of said first beads being coated with a bindingsubstance or being magnetic such that each first bead is specificallyadapted to bind to at least one of said first cells, c) incubating saidfirst beads with said suspension for a period of time effective topermit said first cells to bind to said first beads to form firstbead-first cell complexes, each first bead-first cell complex comprisinga first bead and a first cell, d) separating said first bead-first cellcomplexes from said suspension by filtration through a filter, whereinsaid filter permits effective separation of said first bead-first cellcomplexes from said suspension, and e) examining said separated firstbead-first cell complexes and characterizing said first cells, saidmethod further comprising the step of adding to a preparatory suspensionof said cells a fixative in an amount effective to inhibit phagocytosisof beads and subsequently washing out said fixative prior to saidcontacting in step (b).
 2. The method of claim 1, wherein said fixativeis selected from the group consisting of ethanol, formaldehyde,paraformaldehyde, methanol, glutaraldehyde and mixtures thereof.
 3. Themethod of claim 1, wherein said suspension of cells includes secondcells, said second cells and said first cells being in groups which donot overlap, said method further comprising the steps of: 1) prior tosaid filtration step, adding to said suspension a group of substantiallyidentical second beads, each of said second beads being coated with abinding substance or being magnetic such that each second bead isspecifically adapted to bind to a second cell, 2) incubating said secondbeads with said suspension for a period of time effective to permit saidsecond cells to bind to said second beads to form second bead-secondcell complexes, and 3) simultaneously separating said second bead-secondcell complexes and said first bead-first cell complexes from saidsuspension by filtration.
 4. The method of claim 3, wherein said firstbeads and said second beads are visually distinguishable in size orcolor.
 5. The method of claim 1, wherein said suspension of cellsincludes second cells which are a subset of said first cells, saidmethod further comprising the steps of: 1) prior to said filtrationstep, adding to a suspension comprising said first beads and first cellsa group of substantially identical second beads, each of said secondbeads being coated with a binding substance or being magnetic such thateach second bead is specifically adapted to bind to a second cell, saidfirst beads being distinguishable from said second beads, 2) incubatingsaid second beads with the suspension for a period of time effective topermit said second beads to bind to said second cells to form firstbead-second cell-second bead complexes, each first bead-secondcell-second bead complex comprising a first bead, a second cell, and asecond bead, and 3) separating said first bead-second call-second beadcomplexes from said suspension by filtration.
 6. The method of claim 1,said first beads have a diameter of at least 2 microns but not more than30 microns.
 7. The method of claim 1, comprising the step oftransferring to a glass slide the bead-cell complexes separated by saidfiltration.
 8. The method of claim 1, wherein said binding substance onsaid first beads comprises an antibody.
 9. The method of claim 1,including a step of visually examining said separated first bead-firstcell complexes and visually characterizing said first cells.
 10. Themethod of claim 1, wherein said first beads are plastic microspheres.11. The method of claim 1, wherein said fixative is ethanol.
 12. Amethod of processing cells comprising the steps of: a) providing asuspension of cells in a liquid medium, said cells including first cellsb) contacting a group of first beads with said suspension, each of saidfirst beads being coated with a binding substance or being magnetic suchthat each first bead is specifically adapted to bind to at least one ofsaid first cells, c) incubating said first beads with said suspensionfor a period of time effective to permit said first cells to bind tosaid first beads to form first bead-first cell complexes, each firstbead-first cell complex comprising a first bead and a first cell, d)separating said first bead-first cell complexes from said suspension byfiltration through a filter, wherein said filter permits effectiveseparation of said first bead-first cell complexes from said suspension,and e) examining said separated first bead-first cell complexes andcharacterizing said first cells, said method further comprising the stepof adding to a preparatory suspension of said cells a fixative in anamount effective to preserve said cells for later analysis andsubsequently washing out said fixative prior to said contacting in step(b).
 13. The method of claim 12, wherein said fixative is selected fromthe group consisting of non-crosslinking fixatives.
 14. The method ofclaim 12, wherein said suspension of cells includes second cells, saidsecond cells and said first cells being in groups which do not overlap,said method further comprising the steps of: 1) prior to said filtrationstep, adding to said suspension a group of substantially identicalsecond beads, each of said second beads being coated with a bindingsubstance or being magnetic such that each second bead is specificallyadapted to bind to a second cell, 2) incubating said second beads withsaid suspension for a period of time effective to permit said secondcells to bind to said second beads to form second bead-second cellcomplexes, and 3) simultaneously separating said second bead-second cellcomplexes and said first bead-first cell complexes from said suspensionby filtration.
 15. The method of claim 14, wherein said first beads andsaid second beads are visually distinguishable in size or color.
 16. Themethod of claim 12, wherein said suspension of cells includes secondcells which are a subset of said first cells, said method furthercomprising the steps of: 1) prior to said filtration step, adding to asuspension comprising said first beads and first cells a group ofsubstantially identical second beads, each of said second beads beingcoated with a binding substance or being magnetic such that each secondbead is specifically adapted to bind to a second cell, said first beadsbeing distinguishable from said second beads, 2) incubating said secondbeads with the suspension for a period of time effective to permit saidsecond beads to bind to said second cells to form first bead-secondcell-second bead complexes, each first bead-second cell-second beadcomplex comprising a first bead, a second cell, and a second bead, and3) separating said first bead-second cell-second bead complexes fromsaid suspension by filtration.
 17. The method of claim 12, furthercomprising the step of: a) prior to contacting the first beads with thesuspension, permeabilizing said first cells and incubating said firstcells wit paramagnetic microspheres which are bound to a reactivebiomarker such that said reactive biomarker binds intracellularly insaid first cell, and wherein each first bead is magnetic.
 18. The methodof claim 12, wherein said first beads have a diameter of at least 2microns but not more than 30 microns.
 19. The method of claim 12,further comprising the step of transferring to a glass slide thebead-cell complexes separated by said filtration.
 20. The method ofclaim 12, wherein said fixative is selected from the group consisting ofmethanol and ethanol.